液-质联用法同时测定大鼠血浆中的乌头碱、新乌头碱、次乌头碱及其药动学

Simultaneous determination of aconitine,mesaconitine and hypaconitine in rat plasma by HPLC-MS and its application to pharmacokinetic studies

目的：建立HPLC-MS分析方法同时测定大鼠血浆中的乌头碱、新乌头碱、次乌头碱含量，并用于研究大鼠口服附子煎液后乌头碱、新乌头碱、次乌头碱的药动学。方法：血浆经氨水碱化后用醋酸乙酯进行液液萃取。色谱柱为Alltima C18柱（250mm×4．6mm，5μm），流动相为甲醇-10mmol·L^-1醋酸铵水溶液（75：25）。质谱检测方式为选择性离子监测。选择监测的离子为m／z646．45（乌头碱），m／z632．38（新乌头碱），m／z615．64（次乌头碱）和m／z 336．60（盐酸小檗碱，内标）。结果：血浆中乌头碱在0．05～5μg·L^-1、新乌头碱在0．5～50μg·L^-1、次乌头碱在2．5-250μg·L^-1范围内线性关系良好，定量限为0．05μg·L^-1，血浆中的平均提取回收率高于90％，批内和批间精密度均小于15％。结论：本法灵敏、可靠、简便，适用于乌头碱、新乌头碱、次乌头碱的药动学研究。

OBJECTIVE An HPLC MS assay was established to determine aconitine, mesaconitine and hypaconitine simultaneously in rat plasma and used to investigate the pharmacokinetics of the three alkaloids. METHODS The plasma was alkalized by ammonia water and extracted by ethyl acetate. The separation was carried out on an Alltima C18 column（250mm×4.6mm,5μm）at 35 ℃. The mobile phase was methanol-10 mmol·L^-1 ammonium acetate（75：25）. HPLC-MS was performed in the selected ion monitoring （SIM） mode using target ions at m/z 646.45, m/z 632.38 and m/z 615.64 for the three alkaloids respectively and m/z 336. 60 for the internal standard. A single dose of Radix Aconiti Lateralis Preparata was given to rats orally. Concentrations of aconitine, mesaconitine and hypaconitine in rat plasma were determined by HPLC-MS assay and main pharmacokinetic parameters were estimated. RESULTS The linear calibration curves were obtained in the concentration range of 0. 05- 5μg·L^-1 for aconitine, 0. 5 -50 μg·L^-1 for mesaconitine and 2.5-250μg·L^-1 for hypaconitine. The limits of quantification for the three alkaloids were all 0. 05 ng·mL^-1. The average extraction recoveries were more than 90% in rat plasma samples. The intra day and inter-day precision were less than 15%. CONCLUSION This method is proved to be sensitive, reliable and simple, wbich is suitable for the study of pharmacokinetics of aconitine, mesaconitine and hypaconitine.